The present invention is in the field supramolecular structures that specifically bind to a selected target.
Organized molecular systems are well known in biology and chemistry. For example, pure molecular compounds form crystals, and surface active molecular compounds form monolayers at air-water interphase and vesicles in water. Bilayers of liposomes mimic biological membranes, and biological membranes are good examples of multimolecular organized systems. Viruses, in particular, are highly organized supramolecular assemblies whose complexity surpasses any man-made assembly. Another prime example is the DNA double helix, which is the result of highly selective interaction of two complementary single strand molecules. Man made, or artificial examples of supramolecular systems, include cryptates, i.e., inclusion complexes of macrocyclic receptor molecules, and interrupting two dimensional hydrogen bonded network by a large capping molecule. In these state-of-the-art examples, the structure of all participating molecules are highly specific.
Jean-Marie Lehn has defined supramolecular chemistry as the chemistry beyond individual molecules, i.e., the chemistry of the intermolecular bond. Early work in supramolecular chemistry involved crown ethers and cryptates, compounds based on the interaction of electron pair and ion and possibly additional ion-ion interaction (J.-M. Lehn, Angew. Chem. Int. Ed. Engl. 29 (1990) 1304-1319).
Oligobipyridines form in the presence of suitable metal cations such as copper(II) double-stranded helicates.
Auxiliary groups may be attached into bipyridine units. If these groups are nucleotides they may serve as recognition sites for DNA (U. Koert, M. M. Harding and J.-M. Lehn, Nature (1990) 346:339).
Most previously described hydrogen bonded supramolecules are supramolecular polymers, i.e., periodic supramolecules composed of one or two repeating units. In principle the number of repeating units of polymeric supramolecules may be larger than two but until now nobody has used more than two repeating units. Examples of this class of supramolecules includes the chain-like supramolecule formed by co-crystallization of 1:1 mixture of 2,4,6-triaminopyrimidine and a suitable barbituric acid derivative (J.-M. Lehn, M. Mascal, A. DeCian, J. Fisher, J. Chem. Soc. Chem. Commun. (1990) 479).
Polymeric supramolecules formed from a single unit may also be used. For example, a tubular supramolecule has been formed from a single cyclic peptide (M. R. Ghadiri, J. R. Granja, R. A. Milligan, D. E. McRee and N. Khazanovich, Nature (1993) 366:324-327). These polymeric supramolecules are often simply crystals or mixed crystals in which hydrogen bonding plays a predominant role in structure maintenance. Even, if these supramolecules are stable in solution, their size is variable like that of a conventional polymer.
A step towards controlling supramolecular size and shape has been the use of capping molecules to interrupt the molecular association at the desired point (J. P. Mathias, C. T. Seto, J. A. Zerkowski and G. M. Whitesides in xe2x80x9cMolecular Recognition: Chemical and Biochemical Problems IIxe2x80x9d (Ed. S. M. Roberts) Royal Society of Chemistry). A mixture of he isocyanurate derivative (benzCA2) and trismelamine derivative (trisM3) gives the supramolecule (trisM3)2 (benzCA2)3. This strategy typically produces supramolecules which have xe2x80x98molecular weightxe2x80x99 of 4-10 KDa.
No process exists today for creating large molecular assemblies of deliberately chosen molecules in which the location of the molecules in the assembly can be selected accurately with respect to each other. Nonetheless, a dire need exists for such molecular structures since they could have numerous important medical, chemical and physical applications. These applications include, but are not limited to, supramolecular drugs, drug delivery to target organs, capture of viruses and catalysts, sensors and nanotechnological components.
Polypeptides and proteins, especially enzymes, have been attached to oligonucleotides. A peptide or protein has been used as a tag for an oligonucleotide or oligonucleotide is used as a tag for a polypeptide. Techniques such as ELISA allowed to trace enzymes easier than oligonucleotides, enzymes were used as tags for oligonucleotides. PCR provides for assays of extreme sensitivity. Oligonucleotides are often used as a tag for polypeptides or peptidomimetics, so that the fate of the polypeptide can be followed in vitro or in vivo. Synthesis methods which are used to prepare these conjugates are also useful in this invention. (D. Pollard-Knight, Technique (1990) 3:113-132).
Linear single-stranded tRNA forms branched structures because there are several complementary pieces of the sequence are suitably located. Recently, several two and three dimensional structures have been formed using this principle (Y. Zhang and N. C. Seeman, J. Am. Chem. (1994) 116:1661-1669; N. C. Seeman, J. Theor. Biol. (1982) 99:237-247.). These DNA based supramolecules have been bound together to form active structures. Because several steps are typically needed to create these molecules, the overall synthesis yield can be very low (0.1-1%) because of these steps alone.
Branched pre-mRNA is found in cells. These molecules have highly specific structures in which adenosine is always linked to guanosine. These branched RNAs have been synthesized (T. Horn and M. S. Urdea Nucleic Acid. Res. (1989) 17:6959-6967; C. Sund, A. Fxc3x6ldesi, S.-I. Yamakage and J. Chattopahyaya, Nucleic Acid. Res. (1991) 9-12). The synthesis of branched nucleic acids has been extended to the synthesis of nucleic acid dendrimers (R. H. E. Hudson and M. J. Damha, J. Am. Chem. Soc. (1993) 113:2119-2124).
Oligonucleotide comb and fork structures have been used for analytical purposes (M. S. Urdea, B. Warner, J. A. Running, J. A. Kolberg, J. M. Clyne, R. Sanchez-Pescador and T. Horn (Chiron Corp.) PCT Int. Appl. No. WO 89/03,891 05 May 1989, U.S. application Ser. No. 109,282, 15 Oct. 1987. 112 pp).
All previously known supramolecular structures have some drawbacks. It is of interest to provide novel supramolecular structures that may be adapted for a variety of uses, including disease therapy, diagnostics, assays, and electronics.
The present invention provides several different binding molecule-multienzyme complexes capable of specifically binding to a target of interest. The binding molecule-multienzyme complexes of the invention comprise two or more different effector molecules joined to each other by a joining component, wherein at least one of the effector molecules has the property of binding to a molecular target, i.e. a binding effector molecule, and at least one of the other effector molecules is a therapeutic effector molecule. The joining components for use in the binding molecule-multienzyme complexes of the invention may be of a variety of classes including liposomes, proteins, organic polymers (including dendrimer type polymers). Another aspect of the invention to provide binding molecule-multienzyme complexes in which the joining component is of sufficient length and/or flexibility to permit the therapeutic effector molecules to physically interact with the same target as binding molecule at the same time as binding effector molecule is interacting with the target.
One aspect of the invention relates to binding molecule-multienzyme complexes that are supramolecules formed by at least two supramolecular component molecules. Each supramolecular component molecule comprises at least one effector molecule and at least one nucleic acid chain. At least one of the nucleic acid chains on at least one component molecule of the supramolecules of the invention are complementary to nucleic acid chains on at least one other component, and thus are able to bind the components of the supramolecule by the formation of double stranded nucleic acid chains between the complementary chains. The present invention also provides methods of making the supramolecules of the present invention.
The nucleic acid chains of the supramolecules of the invention are preferably DNA, RNA and may also contain structural analogues of DNA or RNA. Effector molecules that may be used to form the supramolecules include, but are not limited to polypeptides, proteins, lipids, sugars. These effector molecules may impart chemical and physical properties to the supramolecule include, hydrophobicity, hydrophilicity, electron conductivity, fluorescence, radioactivity, biological activity, cellular toxicity, catalytic activity, molecular and cellular recognition and in vivo transport selectivity.
Another aspect of the invention is to provide binding molecule-multienzyme complexes of the invention that may be used to treat or prevent infectious diseases, particularly viral infectious diseases. Binding molecule-multienzyme complexes suitable for the treatment and/or prevention of infectious diseases comprise effector molecules that are antibodies specific for one or more antigen on a viral particle and one or more enzyme capable of catalyzing a reaction that destroys the infectivity of the virus of interest, e.g., hydrolysis of viral coat proteins or viral envelope lipids.
An effector molecule for use in the invention may also be a toxin, such as ricin, which will kill the cell, if the virus is internalized. Another aspect of the invention is to provide binding molecule-multienzyme complexes adapted for the treatment of non-infectious diseases. Binding molecule-multienzyme complexes for the treatment of specific diseases may comprise binding effector molecules specific for certain cells or tissues and effector molecules that serves to directly alleviate a given disease condition.
Another aspect of the invention is to provide binding molecule-multienzyme complexes that expedite the delivery of polynucleotides and other macromolecules into the interior of cells. Such binding molecule-multienzyme complexes are supramolecular structures derived from two or more supramolecular components adapted for the internalization of macromolecules may comprise effector molecules that either alone, or in combination with other effector molecules, on the same or different structure, that are capable of crosslinking receptors on the surface of a cell for transformation.
Another aspect of the invention is to provide binding molecule-multienzyme complexes useful for performing assays for compounds of interest, particularly immunoassays. Supermolecular structures for use in assays typically comprise an effector molecule capable of specifically binding to a compound of interest and a second effector molecule that may capable of producing a detectable signal, e.g., an enzyme, or a second molecule capable of specifically binding to a compound of interest. Another aspect of the invention is to provide assays employing binding molecule-multienzyme complexes of the invention so as provide for the detection and/or quantitation of compounds of interest.
Another aspect of the invention is to provide binding molecule-multienzyme complexes useful for the prevention and treatment of atherosclerosis and related cardiovascular disorders. Binding molecule-multienzyme complexes of the invention useful for the treatment of such diseases may comprise an effector molecule that is an antibody specific for antigens in atherosclerotic plaque.
Another aspect of the invention is to provide method and compositions for the genetic manipulation of cells of interest. The methods and compositions may be used for in vivo and in vivo genetic therapy. The compositions for use in genetic therapy are binding molecule-multienzyme complexes adapted for genetic manipulation. Preferably, such binding molecule-multienzyme complexes adapted for genetic therapy comprise liposomes as joining molecules and further comprise a genetic manipulation complex. Genetic manipulation complexes comprise a polynucleotide for genetic manipulation, a motor protein, and a plurality of site-specific DNA binding proteins. Preferably, the genetic manipulation complex comprises and enzyme with phospholipase activity.